Optimized detector readout for biosensor

ABSTRACT

The present invention provides a biosensor system comprising a light source, a cartridge adapted to be illuminated by said light source, a light detector adapted for detecting a signal originating from the cartridge, an illumination control means adapted to vary the illumination of the cartridge between at least two different states, a means for generating a first oscillation with a first frequency, and a means for generating a second oscillation with a second frequency, wherein the frame rate of the light detector is triggered by the first oscillation and the illumination control means is triggered by the second oscillation.

FIELD OF THE INVENTION

The invention relates to a method of improved readout of an opticaldetector of biosensors and to a system to be used for said method.

BACKGROUND OF THE INVENTION

The demand for biosensors is increasingly growing these days. Usually,biosensors allow for the detection of a given specific molecule withinan analyte, wherein the amount of said molecule is typically small.Therefore, target particles, for example super-paramagnetic label beads,are used which bind to a specific binding site or spot only, if themolecule to be detected is present within the analyte. One knowntechnique to detect these label particles bound to the binding spots isFTIR. Therein, light is coupled into the sample at an angle of totalinternal reflection. If no particles are present close to the samplesurface, the light is completely reflected. If, however, the labelparticles are bound to said surface, the condition of total internalreflection is violated, a portion of the light is scattered into thesample and thus the amount of light reflected by the surface isdecreased. By measuring the intensity of the reflected light with anoptical detector, it is possible to estimate the amount of particlesbound to the surface.

Typically, a photodiode is used as an optical detector. However, adetection by a (CCD) camera or any other multi-pixel system is much moreefficient, since a camera allows for the parallel detection of variousbinding sites or spots. The disadvantage of the use of a camera, though,is that accurate measurements of the intensity of the binding spots isdifficult due to gain- and offset errors in the read-out system.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide animproved optical detector readout for biosensors. This object isachieved with the features of the claims.

The present invention is based on the idea to modulate the amplitude ofthe light coupled into the sample and to simultaneously detect the lightreflected by the sample surface or originating from the sample. Thus,the relative darkening of the binding spot(s) can be measuredaccurately.

The present invention provides a biosensor system comprising a lightsource, a cartridge adapted to be illuminated by said light source, alight detector adapted for detecting a signal originating from thecartridge, an illumination control means adapted to vary theillumination of the cartridge between at least two different states, ameans for generating a first oscillation with a first frequency, and ameans for generating a second oscillation with a second frequency,wherein the frame rate of the light detector is determined, e.g.triggered, by the first oscillation and the illumination control meansis determined, e.g. triggered, by the second oscillation.

The frame rate of the detector being triggered by the first oscillationdoes not imply that the integration time of the detector is determinedby said oscillation as well. The integration time may be eitherdetermined by the detector or its control or by the waveform of saidfirst oscillation. The same holds true for the triggering of theillumination control means by the second oscillation.

Therein the second frequency is preferably equal to one half of thefirst frequency. Thus, the cartridge experiences two differentillumination states, for example, one illumination state, which issubstantially dark, and a second illumination state, which issubstantially bright. Due to the triggering of the light detector by thefirst oscillation alternate images are taken from the dark and thebright state. This allows for an improved image analysis, since saiddark state contains information regarding noise, background,inhomogeneous illumination and the like.

However, other frequency ratios for dark versus bright frames than 1:1are possible as well. This, in particular, depends on the band width ofthe disturbances, i.e. the noise. A ratio of 1:1 combines maximumreduction band-width with maximum number of bright frames. Yet, if onlya slowly varying dark current has to be suppressed, this ratio may be,e.g., 1:10. In that case, most of the frames are bright allowing formore information to be gathered from the binding spots themselves.

The light detector may, in particular, comprise a video or CCD camera.In that case, the (electronic) shutter of the camera may be usedalternatively to modulate the light amplitude between two values insteadof modulating the light source itself. However, this does not allow forbackground light induced before the camera sensor to be suppressed.

But other detectors are conceivable as well, for instance discretephoto-diodes or any suitable multi-pixel system.

The system may further comprise a video processing software foranalyzing the output of the video or CCD camera and optics forprojecting the light reflected by or originating from the cartridge ontothe light detector or camera.

The present invention also relates to a method of detecting relativedarkening at a binding spot of a biosensor cartridge. Said methodcomprises the steps of taking a first image of the binding spot and itssurrounding at a first illumination and analyzing the intensity of saidfirst image at the binding spot I₁(A) and at its surrounding I₁(B). Thena second image of the binding spot and its surrounding at a secondillumination is taken and the intensity of said second image at thebinding spot I₂(A) and at its surrounding I₂(B) is analyzed. Finally,the relative darkening of the binding spot is calculated. Therein, thesecond illumination has a higher intensity than the first illumination.Optionally, the method further comprises the step of identifying thebinding spot.

Preferably, the relative darkening D of the binding spot is calculatedby D=(I₂(A)−I₁(A))/(I₂(B)−I₁(B)). But other ways to calculate therelative darkening are possible as well. For example, the steps ofanalyzing the intensity of the first and second image may be performedpixel-wise.

Of course, neither the biosensor system nor the method of detection islimited to frame-by-frame illumination modulation. Different parts ofthe frame may be illuminated differently. It is, for example,conceivable that the detector progressively scans pixels or regionslike, e.g., horizontal stripes of the image.

The described method may be implemented into different known techniquesto perform bio-sensing. For instance, the amount of label particlesclose to the sensor surface may be measured by frustrated total internalreflection (FTIR). However, the method according to the presentinvention is not limited to any specific sensing technique or sensor.The sensor can be any suitable sensor to detect the presence ofparticles on or near to a sensor surface, e.g. by imaging, fluorescence,chemi-luminescence, absorption, scattering, evanescent field techniques,surface plasmon resonance, Raman, etc.

These and other aspects of the invention will be apparent from andelucidated with reference to the embodiment(s) described hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows a camera picture obtained by FTIR.

FIG. 2 illustrates a camera response for two different gains.

FIG. 3 schematically shows an embodiment of an FTIR system according tothe present invention.

FIG. 4 a depicts a diagram showing the illumination light intensityversus time.

FIG. 4 b depicts a diagram showing the light intensity of region Aversus time.

FIG. 4 c depicts a diagram showing the light intensity of region Bversus time.

DETAILED DESCRIPTION OF EMBODIMENTS

FIG. 1 schematically shows a camera picture obtained by FTIR. Thispicture is obtained by (predominantly) homogeneous illumination of theFTIR surface of a cartridge and projection of the reflected light via anoptical system on a (CCD) camera. Therein, six binding sites or spotsA₁, A₂ can be identified in a region B with a certain backgroundillumination or noise level. The brightness of the binding sites dependson the amount of label particles bound to these sites: The more labelparticles are bound the more light is scattered at the respective regioncausing a decrease of the reflected light. Thus, a dark binding siteindicates a large amount of bound label particles. The relativedarkening D of a binding spot compared to the surrounding area is ameasure for the number of bindings and accordingly for the amount of aspecific molecule within the sample liquid.

However, said relative darkening cannot be measured accurately becauseof gain- and offset errors in the read-out system. This is caused byunknown optical gain in the optical light path, the level ofillumination, gain parameters in the camera (exposure time, shutter,active pixels etc.). Further negative effects are spurious backgroundlight and dark current in the camera.

In a typical FTIR geometry, small signal changes from the labelparticles or beads are detected on a relatively large optical base linesignal. Said optical base line signal originates from the largereflection from the surface of the binding surface. Due to this largeoptical base line signal, gain variations originating from temperatureeffects (drift) in the sensor, in the signal processing and opticallight path will introduce large variations in the detected signal, whichlimits the achievable accuracy and detection limit of the biosensor.This is especially a problem during relatively long-time measurementsfor low target-concentrations.

Moreover, demands on the dynamic range of the detection electronics arequite severe for high-sensitivity applications. Furthermore, spuriouslight sources from ambient and lighting may disturb the measurement.

The camera response, which is the output per pixel versus the appliedlight intensity, can vary between pixels due to manufacturingtolerances. Furthermore the gain depends on many parameters likeexposure time, shutter setting, selected pixels (black-white or color)and is not well defined. FIG. 2 illustrates two different responses of atypical pixel due to different gains. The dynamic range is generallydetermined by the resolution, e.g. 8 bits.

FIG. 3 schematically shows an embodiment of an FTIR system according tothe present invention. An FTIR cartridge 1 is illuminated by a lightsource 2. This may be, e.g., a laser or LED. The incoming light 2 afulfils the condition of total internal reflection and is reflected at asensor surface of the cartridge 1. The reflected light 3 a is detectedat a camera 3, which may be, e.g., a CCD camera. The incoming light 2 agenerates an evanescent wave within the cartridge 1. A portion of saidwave is scattered if an optical inhomogeneity, e.g., a label particle,is present close to the surface of the cartridge. Accordingly, theintensity of the reflected light 3 a detected at camera 3 is varied.

According to the invention, the light source 2 is controlled by anillumination light control means 4. In particular, the intensity of thelight emitted by the light source 2 can be controlled by control means4.

The system further comprises an oscillator 6, which generates a firstoscillation of a first frequency, e.g., 30 Hz. The frame rate of thecamera 3 is determined by said first oscillation. A divider 5 dividesthe first frequency by a factor, preferably 2, in order to generate asecond oscillation with a second frequency. In the exemplary case of thefirst frequency being 30 Hz and the factor being 2, the second frequencywill be 15 Hz. Said second oscillation triggers the illumination lightcontrol means 4.

Thus, the intensity of the illumination is varied with a secondfrequency, e.g. Hz, whereas the frame rate of the camera equals a firstfrequency, e.g., 30 Hz. Accordingly, bright and dark pictures of thereflected light 3 a are taken in an alternating manner. These bright anddark pictures are then analyzed by a software 7, e.g. LabView or anyother suitable software.

Alternatively the (electronic) shutter of the camera 3 may be used tomodulate the light amplitude between two values instead of modulatingthe light source 2 itself. As already mentioned above, this does notallow for a reduction of background light.

FIG. 4 a depicts a diagram showing the varying intensity of theillumination light 2 a versus time. As described above, a dark framewith a low intensity I_(L) (which may be zero) is followed by a brightframe with a high intensity I_(H). Accordingly, the background intensityof region B (cf. FIG. 1) of the pictures taken by camera 3 will vary ina corresponding pattern as shown in FIG. 4 c: If the cartridge isilluminated with low intensity the background intensity B_(L) will below as well. If the cartridge is illuminated with high intensity thebackground intensity B_(H) will be increased as well.

The same holds true for the intensity of a binding spot A (cf. FIG. 1).The alternating intensities A_(L) and A_(H) of a specific binding spotor site are shown in FIG. 4 b. Of course, the absolute values of theseintensities will depend on the specific binding spot as is apparent fromFIG. 1. Nevertheless, the pattern of varying intensity over time issimilar for all binding spots.

Said bright and dark frames are transferred to the video processingsoftware 7, which makes interfacing stable and robust, since the cameraand software are “slave” of the illumination sequence and there is noneed for interface control loops or the like.

The relative average darkening D of the binding spots can easily becalculated according to

$D = {\frac{A_{H} - A_{L}}{A_{H_{t = 0}} - A_{L_{t = 0}}} \cdot \frac{B_{H_{t = 0}} - B_{L_{t = 0}}}{B_{H} - B_{L}} \cdot {{100\lbrack\%\rbrack}.}}$At the start of the assay the cartridge is “white”, hence the averageintensities at t=0 (i.e., the beginning of the assay) fulfil theequation A_(H) _(t=0) −A_(L) _(t=0) =B_(H) _(t=0) −B_(L) _(t=0) . As aresult the relative darkening D of the binding spots reduces to

${D = {\frac{A_{H} - A_{L}}{B_{H} - B_{L}} \cdot {100\lbrack\%\rbrack}}},$where B_(L) and B_(H) are the averaged light values across the totalbackground area B.

This method, however, gives no information of the binding distributionwithin a binding spot as just the average relative darkening iscalculated for each binding spot. Therefore, an improved processing issuggested to calculate the relative darkening per pixel.

When the binding distribution within a binding-spot has to be assessed,there is a need for a gain- and offset corrected image. Hence, for everypixel P in the image the relative darkening D_(P) is calculatedreferenced to the white area according to:

$D_{P} = {\frac{P_{H} - P_{L}}{P_{H_{t = 0}} - P_{L_{t = 0}}} \cdot \frac{B_{H_{t = 0}} - B_{L_{t = 0}}}{B_{H} - B_{L}} \cdot {{100\lbrack\%\rbrack}.}}$In contrast to the method described above, P_(H) _(t=0) −P_(L) _(t=0)≠B_(H) _(t=0) −B_(L) _(t=0) , because the actual pixel values maydeviate from the average.

Obviously, the average relative darkening of the binding spots A is:

${D = \frac{\sum\limits_{i = 1}^{n}D_{P_{i}}}{n}},$which is more accurate in case of non-uniform offset light compared tothe value obtained with the method described above.

The system of the present invention provides several advantages: Noforward sense or monitor diode needed, as is typically the case forFTIR. The achieved results are gain independent. Spurious backgroundlight and camera dark-current are suppressed. And the interfacing tosoftware of the system is easy and robust.

Of course, the method described in the present invention, althoughdescribed with respect to an FTIR sensor, is not restricted to FTIR. Thesensor can be any suitable sensor to detect the presence of particles onor near to a sensor surface, based on any property of the particles,e.g. by reflective or transmissive imaging, fluorescence,chemiluminescence, absorption, scattering, evanescent field techniques,surface plasmon resonance, Raman, etc.

The method according to the present invention can be used with severalbiochemical assay types, e.g. binding/unbinding assay, sandwich assay,competition assay, displacement assay, enzymatic assay, etc. The methodsof this invention are suited for sensor multiplexing (i.e. the paralleluse of different sensors and sensor surfaces), label multiplexing (i.e.the parallel use of different types of labels) and chamber multiplexing(i.e. the parallel use of different reaction chambers). The methodsdescribed in the present invention can be used as rapid, robust, andeasy to use point-of-care biosensors for small sample volumes. Thereaction chamber can be a disposable item to be used with a compactreader, containing the one or more magnetic field generating means andone or more detection means. Also, the methods of the present inventioncan be used in automated high-throughput testing. In this case, thereaction chamber is e.g. a well plate or cuvette, fitting into anautomated instrument.

While the invention has been illustrated and described in detail in thedrawings and foregoing description, such illustration and descriptionare to be considered illustrative or exemplary and not restrictive; theinvention is not limited to the disclosed embodiments. Other variationsto the disclosed embodiments can be understood and effected by thoseskilled in the art in practicing the claimed invention, from a study ofthe drawings, the disclosure, and the appended claims. In the claims,the word “comprising” does not exclude other elements or steps, and theindefinite article “a” or “an” does not exclude a plurality. A singleprocessor or other unit may fulfill the functions of several itemsrecited in the claims. The mere fact that certain measures are recitedin mutually different dependent claims does not indicate that acombination of these measured cannot be used to advantage. Any referencesigns in the claims should not be construed as limiting the scope.

The invention claimed is:
 1. A system comprising: a source for providingan incoming light; a cartridge having a reflective surface and labelparticles causing an optical inhomogeneity, the cartridge is configuredto generate an evanescent wave having a portion scattered due topresence of the label particles in response to illumination by theincoming light; a light detector configured to detect light originatingfrom the cartridge; a controller connected to the source and configuredto control the source to vary an intensity of the incoming light tosequence between at least two different illumination states; anoscillator connected to the controller and the light detector andconfigured to generate a first oscillation having a frequency triggeringa frame rate of the light detector to control detection of the incominglight reflected from the cartridge separately in the at least twodifferent illumination states; a divider connected between theoscillator and the controller and configured to receive the firstoscillation and to generate a second oscillation having a frequency fortriggering the controller; and a processor configured to compare a framecaptured at a first of the at least two different illumination states toa frame captured at a second of the at least two different illuminationstates.
 2. The system according to claim 1, wherein the frequency of thesecond oscillation is determined by dividing the frequency of the firstoscillation by a factor.
 3. The system according to claim 2, wherein thefactor is 2 and the frequency of the second oscillation is one half ofthe frequency of the first oscillation, and the light detector capturestwo frames for each of the two different illumination states.
 4. Thesystem according to claim 1, wherein the first illumination state issubstantially dark and the second illumination state is substantiallybright, the first illumination state enabling the light detector tocapture a dark picture frame including low background intensity and thesecond illumination state enabling the light detector to capture abright picture frame including high background intensity.
 5. The systemaccording to claim 4, wherein the detection of the frames from the atleast two different illumination states is controlled in an alternatingmanner.
 6. The system according to claim 4, wherein the processor isfurther configured to utilize the comparison between captured frames togenerate a gain and offset corrected image.
 7. The system according toclaim 4, wherein the processor is further configured to calculate arelative darkening for each pixel in the dark and bright pictures. 8.The system according to claim 7, wherein the processor is furtherconfigured to calculate an average darkening to generate a gain andoffset corrected image.
 9. The system according to claim 1, wherein thelight detector comprises at least one of a video camera and a CCD cameraand is a slave of the illumination sequence.
 10. The system according toclaim 1, wherein the processor is further configured to determine arelative darkening due to the label particles based on the comparisonbetween captured frames and within captured frames.
 11. The systemaccording to claim 1, further comprising optics for projecting the lightoriginating from the cartridge onto the light detector.
 12. The systemaccording to claim 1, wherein the light detector comprises at least oneof a video camera and a CCD camera that directly receives the lightoriginating from the cartridge.
 13. The system according to claim 1,wherein the processor is further configured to to generate a gain andoffset corrected image, and to determine a darkening due to the labelparticles as a ratio of dark portions of the corrected image tosurrounding portions of the corrected image.
 14. A detection systemcomprising: a source for generating an incoming light; a cartridgehaving a reflective surface and label particles causing an opticalinhomogeneity, the cartridge is configured to generate an evanescentwave having a portion scattered due to presence of the label particlesin response to illumination by the incoming light; a CCD cameraconfigured to detect the light originating from the cartridge; acontroller connected to the source and configured to control the sourcefor generating an illumination sequence of the incoming light havingalternating low light and high light intensity states; an oscillatorconnected to the CCD camera and the controller for generating a firstoscillation to control a frame rate of the CCD camera to separatelycapture a frame for the low light intensity state and the high lightintensity state and to control the frequency of the illuminationsequence to generate the alternating low light and high light intensitystates; a divider connected between the oscillator and the controller,the divider generating a second oscillation for triggering thecontroller; and a processor configured to compare a frame captured atthe high light intensity state to a frame captured at the low lightintensity state.
 15. The system according to claim 14, wherein theprocessor is further configured to correct for gain and offset in eachcaptured frame by referencing the high light intensity state against thelow light intensity state.
 16. The system according to claim 14, whereinthe processor is further configured to to generate a gain and offsetcorrected image, and to determine a darkening due to the label particlesas a ratio of dark portions of the corrected image to surroundingportions of the corrected image.